Dear Navid,

Hi.

HeLa cells grow very rapidly and efficiently and they are according to my experience, they are very resistant agains various physical stresses.

However, several factors might affect their viability and growth rate. If you seed your cells and they attach but do not replicate, or their morphology is abnormal (like they are too thin, or they form clusters and islands) please make sure:

1. Your culture media is complete and is not lacking any additive (FBS, L-Glutamine, Glucose, etc).

2. Your seeding density is appropriate. Low density seeding migh hamper cell growth.

3. Your cells are not contaminated with mycoplasma or viruses (CMV for example)

4. You are using new and high-quality cell culture vessels.

5. Your incubator is calibrated (in terms of CO2, temperature, humidity).

6. During trypsinization, you do not keep them in trypsin or room temperature for a prolonged time, or pipetting them very harshly.

7. Your centrifuge speed does not exceed 130g (approx. 1500 RPM for large benchtop centrifuges)

8. You perform the centrifugation in RT

In general, you must always be nice to your cells during splitting. But you must be fast too.

On the other hand, if your cells do not attach at all after seeding, always check their viability before seeding. If you have viability below 75%, you must find the reason. It can be any of above. In this case, you have check them individually.

I suggest consulting with your colleagues in your lab (preferably those that use same equipment and materials to culture HeLa) and see how their cell behaviuor differ from yours.

All the best and good luck,

Mohammad

Thanks dear @Mohammad Pirouzfar.

I appreciate you to share your valuable knowledge and experience.

You mentioned essential points I have never found in talking and consulting with expert cell culture technicians.